Systems and methods using nuclear magnetic resonance (nmr) spectroscopy to evaluate pain and degenerative properties of tissue

ABSTRACT

NMR spectroscopy is performed on intervertebral disc tissue. Extent of degeneration is determined based on the NMR spectroscopy. Correlation between NMR spectral regions and at least one of tissue degeneration and pain are made. Accordingly, NMR spectroscopy is used to determine location and/or extent of at least one of degeneration or pain associated with a region of tissue, such as for example in particular disc degeneration, or discogenic pain. NMR spectral peak ratios, such as between N-Acetyl/cho and cho/carb, are readily acquired and analyzed to predict degree of tissue degeneration and/or pain for: tissue samples using HR-MAS spectroscopy; and larger portions of anatomy such as joint segments such as a spine, using clinical 3T MRI systems with surface head or knee coils; and tissue regions such as discs within spines of living patients using 3T MRI systems with a surface spine coil, thus providing a completely non-invasive diagnostic toolset and method to image and localize degeneration and/or pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application serialnumber 13/680,612 filed on Nov. 19, 2012, incorporated herein byreference in its entirety, which is a continuation of U.S. patentapplication Ser. No. 11/829,847 filed on Jul. 27, 2007, now U.S. Pat.No. 8,344,728, incorporated herein by reference in its entirety, whichis a 35 U.S.C. § 111(a) continuation-in-part of PCT internationalapplication serial number PCT/US2006/003036 filed on Jan. 30, 2006,incorporated herein by reference in its entirety, which claims priorityto, and the benefit of, U.S. provisional patent application Ser. No.60/737,110, filed on Nov. 15, 2005, incorporated herein by reference inits entirety, and which claims priority to, and the benefit of, U.S.provisional patent application Ser. No. 60/648,241, filed on Jan. 28,2005, incorporated herein by reference in its entirety. Priority isclaimed to each of the foregoing applications.

This application is also related to PCT International Publication No. WO2006/081471 published on Aug. 3, 2006 and republished on Aug. 23, 2007,incorporated herein by reference in its entirety. This application isalso related to U.S. Patent Application Publication No.US-2008-0039710-A1 published on Feb. 14, 2008, incorporated herein byreferenced in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under R01-AG17762 andR21-AR51048 awarded by National Institutes of Health. The Government hascertain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN COMPUTER PROGRAMAPPENDIX

Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

A portion of the material in this patent document is subject tocopyright protection under the copyright laws of the United States andof other countries. The owner of the copyright rights has no objectionto the facsimile reproduction by anyone of the patent document or thepatent disclosure, as it appears in the United States Patent andTrademark Office publicly available file or records, but otherwisereserves all copyright rights whatsoever. The copyright owner does nothereby waive any of its rights to have this patent document maintainedin secrecy, including without limitation its rights pursuant to 37C.F.R. § 1.14.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention pertains generally to imaging of tissues associated withskeletal joints, and more particularly to identifying and/orcharacterizing medical conditions associated with skeletal joints, pain,or both. Still more specifically, it relates to using nuclear magneticresonance (NMR) spectroscopy to identify, localize, and/or characterizechemical, molecular, structural, or other signatures related to medicalconditions in tissues, such as degradation or pain associated withskeletal joints (for example spine).

2. Description of Related Art

Intervertebral disc degeneration (IVDD) is a leading cause of lumbarspine related lower back pain, a common medical problem that affects 60to 80% of aging Americans. The intervertebral disc is a flexiblefibrocartilaginous structure that supports forces and facilitates spinalmovement. Healthy discs consist of three specific tissue components: 1)the annulus fibrosus, a collagenous region tightly packedcircumferentially around the periphery of the disc which allows forpliability; 2) the nucleus pulposus, a hydrated, proteoglycan gellocated at the center of the disc, which when compressed expandsradially and braces the annulus fibrosus to maintain stiffness andprevents the annulus from buckling under compression; and 3) acartilaginous end-plate that separates the nucleus from the adjacentvertebral bone.

Disc degeneration is characterized by a complex series of physical andchemical degradative processes. The extent or severity of IVDD is mostcommonly described clinically using the Thompson Grading Scale, wherefollowing a set of parameters, a x-ray radiographic inspection of thedisc is conducted and the gross morphology is used to determine theextent of degeneration. One research group has concluded that changes tothe mechanical properties of the intervertebral disc suggest a shiftfrom a “fluid like” behavior to a more “solid like” behavior withdegeneration. Fixed charge density (FCD) and the biochemical environmentof the surrounding water have also been shown to greatly influencedegeneration; as highly charged proteoglycans attract water and causethe tissue to swell, disc pressurization and spinal load support aredirectly affected. Differences in the Thompson Grade are reflected bychanges in the concentrations of constituents such as collagen andproteoglycans in both the annulus and nucleus. It has been proposed thatbiochemical degradation, upregulation of genes associated with collagenmatrix degradation, and the cumulative effect of mechanical loading, allstimulate the degenerative disc process.

Identification and characterization of disc degeneration thus involves awide array of technological developments and efforts over many years.Yet, an adequate, repeatable, non-invasive system and method tocharacterize factors related to pain, pain generation, or discdegeneration has yet to be provided as a useful medical tool.

It is also well appreciated in current medical practice that pain is aremarkably difficult phenomenon to diagnose and localize. This is inparticular the case with respect to skeletal joint pain, and inparticular back pain. Whereas certain targeted pain relief therapies maybe made available, such as directed energy sources to locally ablatepainful nociceptive nerves, the indentification and localization ofwhere to treat is a critical pacing item that often falls well short ofproviding the requisite specificity. As a result, the ability tosuccessfully target such therapies in overall pain management isextremely challenging at best.

Degenerative disc disease, while a predominant cause of debilitatingback pain, is however only one example of medical conditions in direneed for better tools and methods to characterize and localize thecondition in order to appropriately direct therapies. Chronic back pain,for example, may result from several underlying root causes. Thesecauses include, for example, vertebral compression fractures,degenerative disc disease, and disc herniation. In addition, other jointpain, such as of the spine or other skeletal joints (e.g. knuckles,ankles, knees, hips, shoulders, wrists, elbows) may also be the resultof many different underlying causes (or combinations of them), and mayalso be very difficult to localize sufficiently to direct localizedtherapies. Pain associated with any or all of these joints may belocated at the connective or cushioning tissue of the joint itself (e.g.the disc for spinal joints), or within the bone, or at transitionalareas (e.g. the end-plates of vertebral bodies bordering discs).

A substantial need exists for improved non-invasive tools and methodsfor identifying and characterizing the degradation of tissues in thebody. This is in particular the case with respect to skeletal joints, inparticular intervertebral joints of the spine, and further in particularin and around the intervertebral discs themselves.

A substantial need also exists for improved non-invasive tools andmethods for identifying, characterizing, and/or localizing pain withinthe body. This is also in particular the case with respect to skeletaljoints, in particular intervertebral joints of the spine, and further inparticular in and around the intervertebral discs themselves.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present disclosure provides a medical diagnosticsystem with a non-invasive imaging modality that is adapted to provideuseful information that is indicative of a degree of a property of aregion of tissue based upon a chemical signature of a factor associatedwith that property.

Spectra obtained using the NMR spectroscopic method are very similar forsamples taken from annular and nuclear regions of discs. Visuallyapparent changes are observed in the spectra of the annular and nuclearsamples from discs with increasing Thompson grade. Area ratios of theN-Acetyl to choline regions and the choline to carbohydrate regions ofthe spectra allow for discrimination between discs of increasingThompson grade with minimal overlap of individual ratios.

According to one mode of this aspect, the property is associated withpain.

According to another mode of this aspect, the property is associatedwith tissue degeneration.

According to another mode of this aspect, the system provides usefulinformation that is indicative of a property associated with a skeletaljoint.

According to another mode of this aspect, the system provides usefulinformation that is indicative of a property associated with anintervertebral joint.

According to another mode of this aspect, the system provides usefulinformation that is indicative of a property associated with anintervertebral disc.

According to another mode, the system provides useful informationindicative of a degree of degradation of an intervertebral disc.

According to another mode, the system provides useful informationindicative of a location where pain is being experienced in a patient.

According to another mode, the system provides the useful informationbased at least in part upon an NMR spectrum of the region of tissue.

Another aspect described hereunder is a medical diagnostic systemconfigured to provide useful information that is indicative of aproperty of a first region of tissue based upon a magnetic resonance(NMR) spectrum of the first region, and wherein the property isassociated with at least one of tissue degeneration and pain.

Another aspect described hereunder is a medical diagnostic systemconfigured to provide useful information that is indicative of aproperty of a first region of tissue based upon a nuclear magneticresonance (NMR) spectrum of the first region, and wherein the propertyis associated with tissue degeneration.

Another aspect described hereunder is a medical diagnostic systemconfigured to provide useful information that is indicative of aproperty of a first region of tissue based upon a nuclear magneticresonance (NMR) spectrum of the first region, and wherein the propertyis associated with pain.

Another aspect described hereunder is a system for identifying orcharacterizing a property of a first region of tissue associated with askeletal joint. This aspect includes a processor that is configured toprocess data related to an NMR spectrum of the tissue in a manner thatprovides useful information that is indicative of the property in thefirst region of tissue.

Another aspect described hereunder is a system for identifying orcharacterizing a property of a first region of tissue associated with askeletal joint. This particular aspect includes an NMR spectroscopysystem that is configured to provide an NMR spectrum of the first regionof tissue and to provide data related to the spectrum in a form that isprocessable to provide useful information that is indicative of theproperty in the first region of tissue.

Another aspect described hereunder is a system for identifying orcharacterizing a property of a first region of tissue associated with askeletal joint. This aspect includes a substantially non-invasiveimaging modality that is configured to provide imaging data related tothe first region of tissue in a form that is processable to provideuseful information that is indicative of the property in the firstregion of tissue.

Another aspect described hereunder is a system for identifying aproperty of at least a first region of an intervertebral disc. Thisaspect includes an NMR spectroscopy system adapted to provide datarelated to an NMR spectrum of the first region of tissue in a form thatis processable to provide useful information that is indicative of theproperty in the first region of tissue.

Another aspect described hereunder is a system that provides usefulinformation for diagnosing a property of a first region of tissue thatis associated with tissue degeneration or pain. This aspect includes acomputer readable software program in computer readable media form andthat is configured to process data from a nuclear magnetic resonance(NMR) spectrum taken from the first region of tissue. The program isfurther configured to provide the useful information from the processeddata.

Another aspect of the invention is a system for identifying,characterizing, and/or localizing a property of tissue associated with askeletal joint. Such aspect may further include any one or more of thevarious aspects, modes, embodiments, variations, or features hereinshown or described, or combinations thereof.

According to one mode of this aspect, the system is adapted to provideinformation indicative of a degree of a property of at least a portionof an intervertebral disc.

Another aspect is a system for identifying or characterizing a propertyof tissue associated with a skeletal joint in a patient. This includesat least one of: a processor that is configured to process informationrelated to NMR spectroscopy of the tissue in a manner that is adapted toallow a degree of the property to be identified or characterized; an NMRspectroscopy systems that is configured to provide spectroscopicinformation related to the tissue such that a degree of the property maybe identified or characterized; or a substantially non-invasive imagingmodality that is configured to provide information regarding the tissuesuch that a degree of the property may be identified or characterized.Or, the system may include a combination of two or more of theforegoing.

According to one mode of this aspect, the information is related to adegree of a property of at least a portion of an intervertebral disc.

Another aspect of the invention is a system for characterizing at leasta portion of an intervertebral disc with respect to a degree of aproperty of that disc. This system includes an NMR spectroscopy systemadapted to capture a spectrum related to the portion. The spectrumprovides information that is useful to indicates at least in part thedegree of the property.

According to one further embodiment of the foregoing aspects and modes,the respective system is adapted to produce the information based oneither or both of an annular portion or a nucleus portion of theintervertebral disc.

According to another embodiment, the system is adapted to display acurve related to the spectrum, and a portion of the curve provides theinformation.

According to another embodiment, the information is adapted todistinguish a degree of degradation of the disc. According to one highlybeneficial further embodiment, the information is adapted to distinguishas to the degree of degradation by reference to a Thompson scale.

According to another embodiment, the property comprises at least one ofpain, or at least one factor that correlates with pain.

According to another embodiment, the information is related to ratios ofthe resonances in the N-acetyl to choline regions, and choline tocarbohydrate regions of the spectra.

According to another embodiment, the information is related tochondroitan sulfate, or a metabolite or degradation product thereof.

According to another embodiment, the information relates to at least oneof T₁ and T₂ relaxation times of chemical constituents of disc spectra.

According to another embodiment, the property comprises at least one ofa degree of dehydration of the disc, a degree of breakdown of aproteoglycan matrix of the disc, and a degree in a breakdown of acollagen matrix.

According to another embodiment, the system further includes a protonhigh resolution magic angle spinning spectroscopy system that is adaptedto produce the information.

Another aspect of the invention is a method for identifying orcharacterizing a property of tissue associated with a skeletal joint.One or more of the foregoing method aspects, modes, embodiments,variations, or features herein described, or combinations thereof, maybe employed to advance this method.

One further mode of this aspect further includes providing informationindicative of a degree of a property of at least a portion of anintervertebral disc.

Another aspect is a method for identifying or characterizing a propertyof tissue associated with a skeletal joint in a patient, and includes atleast one of the following steps: processing information related to NMRspectroscopy of the tissue in a manner that is adapted to allow a degreeof the property to be identified or characterized; providingspectroscopy information from an NMR spectroscopy system and that isrelated to the tissue such that a degree of the property may beidentified or characterized; or providing information regarding thetissue from a substantially non-invasive imaging modality with respectto the tissue and such that a degree of the property may be identifiedor characterized. Or a combination of one or more of the foregoing maybe used.

One mode of this aspect includes determining a degree of a property ofat least a portion of an intervertebral disc based upon the information.

Another aspect of the invention is a method for characterizing at leasta portion of an intervertebral disc with respect to a degree of aproperty thereof, and includes capturing a spectrum related to theportion using an NMR spectroscopy system. The spectrum providesinformation that indicates at least in part the degree of the property.

According to one embodiment of the various method aspects and modes justdescribed, the information produced is based on either or both of anannular portion or a nucleus portion of the intervertebral disc.

In another embodiment, a curve is displayed that is related to thespectrum, and wherein a portion of the curve provides the information.

Another embodiment includes distinguishing a degree of degradation ofthe disc based upon the information. A still further embodiment includesdistinguishing the degree of degradation of the disc in relation to aThompson grade based upon the information.

Another embodiment includes correlating the disc with degree of pain, orat least one factor that correlates with pain, based upon theinformation.

According to another embodiment, the information is related to a ratioof at least one of the resonances in the N-acetyl to choline regions,and choline to carbohydrate regions, of the spectra.

According to another embodiment, the information is related tochondroitin sulfate, or a metabolite or degradation product thereof.

According to another embodiment, the information relates to at least oneof T₁ and T₂ relaxation times of chemical constituents of disc spectra.

According to another embodiment, the property relates to at least one ofa degree of dehydration of the disc, a degree of breakdown of aproteoglycan matrix of the disc, and a degree in a breakdown of acollagen matrix.

Another embodiment includes producing the information at least in partusing a proton high resolution magic angle spinning spectroscopy system.

Further to various of the aspects described hereunder, additional modesinclude beneficial non-invasive analysis of tissue properties based uponat least one of lactate-related, proteoglycan-related, orcollagen-related chemical signatures recognized within the tissue.Particular beneficial modes include, for example, comparing ratios ofrecognized features of these signatures, and in one highly beneficialembodiment are based upon NMR resonances of such factors. Of furtherbenefit as provided in the present embodiments, comparisons of two ormore such resonances, such as for example in particular their peaks orother features indicating the extent of their presence, are made toprovide distinguishing results that indicate a degree of a particularcondition in the subject tissue (such as for example extent ofdegeneration or pain).

One additional particularly beneficial mode of the aspects providedhereunder includes use of NMR spectroscopy. In one embodiment, suchspectroscopy involves equipment operating at above 8 Tesla, and infurther embodiment at between about 11 and 12 Tesla. In anotherembodiment, the equipment operates at between about 4 and 10 Tesla, suchas in particular at about 7 Tesla. In another embodiment, it operates atbetween about 2 and 4 Tesla, such as at about 3 Tesla. In anotherembodiment, it operates at below 3 Tesla, such as for example at about1.5 Tesla. In this regard, 3T MRI systems are considered to providesubstantial benefit for spectroscopic imaging & diagnosis of tissueregions within human patients, such as skeletal joints and in particularspinal joints and intervertebral discs. For lower Tesla equipment, stillfurther embodiments contemplate using pass filter augmentation,amplification, or gain of signals falling within particular rangestargeted, such as certain particular peak resonant frequencies targetedas recognized signatures correlating with certain factors to be examinedfor the intended non-invasive diagnosis.

In additional embodiments, local coils may be used in MRI systems forenhanced magnetic resonance data acquisition. For example, local coilssimilar to those previously disclosed and developed for head and neckimaging may be used around skeletal joints for purposes of acquiringdata useful according to the present aspects of this disclosure. In oneparticular embodiment, a local spine coil is used.

Particular beneficial embodiments of the present aspects include usefulanalysis of ratios between regions of NMR spectra that representdifferent chemical constituents in tissue regions being evaluated. It isappreciated that while using such ratios, and in particular certainspecific ratios herein described, the individual components of suchratios representing individual spectral regions associated any one of anumber of chemical constituents, and in particular those hereindescribed with some specificity, are also considered useful data underthe broad aspects and modes and thus represent further embodimentscontemplated hereunder.

Another aspect of the present disclosure evaluates N-Acetyl/cho NMRspectral peak ratios tissue, with decreased values providing a measureof tissue degeneration useful in patient diagnosis. In a further mode,extent of localized pain is diagnosed based upon such evaluation. Inanother further mode the tissue evaluated is intervertebral disc tissue,and according to one embodiment includes at least a portion of a nucleuspulposus.

Another aspect of the present disclosure evaluates N-Acetyl/cho NMRspectral peak ratios in tissue, with increased values providing ameasure of degeneration useful in patient diagnosis. In a further mode,extent of localized pain is diagnosed based upon such evaluation. Inanother further mode the tissue evaluated is intervertebral disc tissue,and according to one embodiment includes at least a portion of a nucleuspulposus.

Another aspect of the present disclosure evaluates choline-related NMRspectral peaks in tissue, with increased values providing a measure ofdisc degeneration useful in patient diagnosis. In a further mode, extentof localized pain is diagnosed based upon such evaluation. In anotherfurther mode the tissue evaluated is intervertebral disc tissue, andaccording to one embodiment includes at least a portion of a nucleuspulposus.

Another aspect evaluates one or more NMR spectral peak ratios in atarget tissue region, compares those values against similar NMR spectralpeak ratios in other adjacent or reference tissue, and determines extentand localization of tissue degeneration and/or pain based upon suchcomparison.

Each aspect, mode, embodiment, variation, or feature herein described isconsidered independently beneficial without requiring combination withthe others. However, such further combinations and sub-combinationsthereof are also considered yet further beneficial independent aspectsinvention.

Further aspects of the invention will be brought out in the followingportions of the specification, including without limitation as presentedin the claims, and wherein the detailed description is for the purposeof describing exemplary and preferred embodiments of the inventionwithout necessarily placing limitations thereon, though such preferredembodiments may be described as providing particularly valuable benefitsand uses.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The invention will be more fully understood by reference to thefollowing drawings which are for illustrative purposes only:

FIGS. 1X, 1Y, 1Z show a representative 1-D HR-MAS spectra acquired aThompson Grade 1 disc (FIG. 1X) from the annulus fibrosus region(spectra at FIG. 1Y) and the nucleus pulposus region (spectra located atFIG. 1Z).

FIGS. 2X, 2Y, 2Z show representative 1-D HR-MAS spectra acquired for aThompson Grade 3 disc (FIG. 2X) from the annulus fibrosus region (FIG.2Y) and the nucleus pulposus region (FIG. 2Z). Resolvable peaks include:A:isoleucine, leucine, valine; B:lactate, isoleucine; C:alanine;D:isoleucine, leucine; E:lysine, leucine; F:N-Acetyl resonance ofchonroitin sulfate; G:glutamine; H:glutamate, proline; I:glutamine,hydroxyproline; J:lysine; K:choline; L:phosphocholine; M:hydroxyproline;N:glycine; O:C-H resonances of chondroitin sulfate; P:ethanoloamine; thebracketed region indicates the C-H resonances of chondroitin sulfate.

FIGS. 3X, 3Y, 3Z show representative 1-D HR-MAS spectra acquired for aThompson Grade 5 disc (FIG. 3X) from the annulus fibrosus region (FIG.3Y) and the nucleus pulposus region (FIG. 3Z). Resolvable peaks include:A:isoleucine, leucine, valine; B:lactate, isoleucine; C:alanine;D:isoleucine, leucine; E:lysine, leucine; F:N-Acetyl resonance ofchonroitin sulfate, Proline, glutamate; G:glutamine; H:glutamate,proline; I:glutamine, hydroxyproline; J:lysine; K:choline; L:phosphocholine; M: hydroxyproline; N:glycine; O:C-H resonances ofchondroitin sulfate; P:ethanoloamine.

FIGS. 4A-4D show, as described in Table 1, the graphical representationof the distribution of integrated N-Acetyl/Cho (FIG. 4A) and Cho/Carb(FIG. 4B) of the annulus fibrosus as well as N-Acetyl/Cho (FIG. 4C) andCho/Carb (FIG. 4D) of the nucleus pulposus with respect to ThompsonGrade. Cho/Carb shows the largest statistical significance.

FIG. 5A shows a rotor synchronized adiabatic TOCSY spectrum of healthydisc material, with an 80 ms mixing time. The horizontal axis is the sumof projections and the vertical axis is a high-resolution 1-D spectrum.The three-letter amino acid code was used to designate amino acidcrosspeaks.

FIG. 5B shows a rotor synchronized adiabatic TOCSY spectrum ofdegenerate disc, with an 80 ms mixing time. In the degenerate spectrumthere is an increase in signal in the amino acids as well as cholinecontaining compounds, which are not present in the healthy spectrum.

FIG. 6A shows a graphical representation of the average spin-latticerelaxation times of the following compounds: 0.9 ppm: Isoleucine,Leucine, and Valine, 1.32 ppm: Lactate, 1.49 ppm: Alanine, 2.04 ppm:N-Acetyl moiety of Chondroitin sulfate, 3.21 ppm: Choline containingcompounds, 3.67 ppm: C-H of the carbohydrate residue associated with theChondroitin sulfate polymer.

FIG. 6B shows a graphical representation of the average spin-spinrelaxation times of the previously mentioned compounds. (*) Denotespeaks with very large standard deviations due to low signal intensity.

FIGS. 7A, 7B show exemplary spectra taken from an experiment performedon certain intervertebral discs using NMR spectroscopy.

FIGS. 8A and 8B show a T2-weighted MRI image of an ex-vivo bovine spinespecimen, and corresponding NMR spectrum of a voxel region in a discnucleus of the spine, respectively, according to certain presentembodiments.

FIGS. 9A and 9B show similar respective images for the the ex-vivobovine spine featured in FIGS. 8A-B, taken at a first time intervalafter Papain injection into the disc evaluated.

FIGS. 10A and 10B show similar respective images for the the ex-vivobovine spine featured in FIGS. 8A-B, taken at a second time intervalafter Papain injection into the disc evaluated.

FIGS. 11A and 11B show similar respective images for the the ex-vivobovine spine featured in FIGS. 8A-B, taken at a third time intervalafter Papain injection into the disc evaluated.

FIGS. 12A and 12B show a T2-weighted MRI image of another ex-vivo bovinespine specimen, and corresponding NMR spectrum of a voxel region in adisc nucleus of the spine, respectively, according to certain presentembodiments.

FIGS. 13A and 13B show similar respective images for the the ex-vivobovine spine featured in FIGS. 12A-B, taken at a first time intervalafter Papain injection into the disc evaluated.

FIGS. 14A and 14B show similar respective images for the the ex-vivobovine spine featured in FIGS. 12A-B, taken at a second time intervalafter Papain injection into the disc evaluated.

FIGS. 15A and 15B show similar respective images for the the ex-vivobovine spine featured in FIGS. 12A-B, taken at a third time intervalafter Papain injection into the disc evaluated.

FIGS. 16A and 16B show a T2-weighted MRI image of a cadaveric spinespecimen, and corresponding NMR spectrum of a voxel region in a discnucleus of the spine, respectively, according to certain presentembodiments.

FIGS. 17A and 17B show a T2-weighted MRI image of another cadavericspine specimen, and corresponding NMR spectrum of a voxel region in adisc nucleus of the spine, respectively, according to certain presentembodiments.

FIGS. 18A and 18B show a T2-weighted MRI image of another cadavericspine specimen, and corresponding NMR spectrum of a voxel region in adisc nucleus of the spine, respectively, according to certain presentembodiments.

FIGS. 19A and 19B show a T2-weighted MRI image of another cadavericspine specimen, and corresponding NMR spectrum of a voxel region in adisc nucleus of the spine, respectively, according to certain presentembodiments.

FIGS. 20A and 20B show a T2-weighted MRI image of another cadavericspine specimen, and corresponding NMR spectrum of a voxel region in adisc nucleus of the spine, respectively, according to certain presentembodiments.

FIGS. 21A and 21B show a T2-weighted MRI image of a portion of a humanpatient's spine, and corresponding NMR spectrum of a voxel region in adisc nucleus of the spine, respectively, according to certain presentembodiments.

DETAILED DESCRIPTION OF THE INVENTION

Referring more specifically to the drawings, for illustrative purposesthe present invention is embodied in the apparatus generally shown inFIG. 1X through FIG. 6B and Table 1, and as further developed accordingto certain particular modes as reflected in FIGS. 7A-B and Table 2. Itwill be appreciated that the apparatus may vary as to configuration andas to details of the parts, and that the method may vary as to thespecific steps and sequence, without departing from the basic conceptsas disclosed herein.

The ability to characterize disc degeneration in regard to particularmaterial or chemical constituents using NMR spectroscopy is hereindisclosed. Non-invasive correlations between NMR spectra indicia andThompson grade are made, yielding tremendous benefit to various uses inmedicine and research. The present invention is highly beneficial withrespect to providing a non-invasive ability to identify and characterizemarkers associated with the particular state or locality of discdegeneration, and in further particular relation to localization of painor pain generating factors.

Various aspects, modes, embodiments, variations, and features of thepresent invention will be made clear by reference to one or moreexperimental studies performed, and accompanying discussion, as providedby way of one or more examples immediately below.

EXAMPLE 1

1. Overview

The goal of this study was to determine the ability of high-resolutionmagic angle spinning (HR-MAS) NMR spectroscopy to distinguish differentstages of intervertebral disc degeneration. 17 discs were removed fromhuman cadavers and analyzed using one- and two-dimensional (TOCSY) ¹HHR-MAS spectroscopy, and T₁ and T₂ relaxation time measurements todetermine the chemical composition and changes in chemical environmentof discs with increasing levels of degeneration (Thompson grade).Significant findings include that spectra were very similar for samplestaken from annular and nuclear regions of discs and that visuallyapparent changes were observed in the spectra of the annular and nuclearsamples from discs with increasing Thompson grade. Area ratios of theN-Acetyl to choline regions, and choline to carbohydrate regions of thespectra allowed for discrimination between discs of increasing Thompsongrade with minimal overlap of individual ratios. Changes in T₁ and T₂relaxation times of the chemical constituents of disc spectra seemed toreflect both changes in dehydration of the disc and the degree ofbreakdown of the proteoglycan and collagen matrices with increasingThompson grade. The results of this study support the using of in vivospectroscopy for detecting chemical changes associated with discdegeneration.

Several in vivo MRI studies have been performed in an attempt to bettercharacterize IVDD. T₁ and T₂ weighted MRI has been used to analyze thestructure of intervertebral discs. A decrease in T₂-weighted signalintensity with increased lumbar disc degeneration has been alleged. T₁values of water in degraded cartilage decrease significantly in sampleswith degeneration. Changes have been allegedly observed in T₂ relaxationtimes of water with degeneration of articular cartilage as well.Diffusion weighted imaging has also been used to study disc andcartilage, showing a decrease in water content as a correlate to adegenerative state. In MRI of the cervical spine, as age increases,dehydration occurs more evenly across all discs. One research groupspeculates that this is due to a more uniform degeneration than that dueto injury or recurrent stress. These MR imaging findings increase theaccuracy of a gross morphological grading system, but degenerativestates could be more effectively and quantitatively measured using amethod based on measurement of the chemical constituents detectablethrough in vivo spectroscopy.

There is currently a great need for non-invasive techniques to bettercharacterize the metabolic composition of intact disc tissue in vivo.Conventional methods for determining chemical composition require theextraction of proteins through biochemical means, which in turn destroythe tissue and prevent further study (e.g., biological assays ormechanical tests). HR-MAS NMR spectroscopy is a non-destructivetechnique that has been successfully used to characterize thecomposition of various intact biological tissues. Cartilage degenerationhas been modeled using collagenases which degrade bovine nasalcartilage, and the degradation products have been studied usinghigh-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Thisallowed the amino acid products of the collagen triple helix to becompared to the natural degradation of bovine tissue and provided amodel of human tissue degradation. However, the differing levels ofbiochemical and mechanical degradation associated with varying degreesof intermediary degradation are still characterized using a singleThompson Grade, which underscores the need for a more descriptivegrading scale than the current method.

The purpose of this study was to demonstrate use of HR-MAS spectroscopyto assess the chemical changes associated with intervertebral discdegeneration. Suitable modifications and adaptations of these HR-MAStools and methods may thus be made in order to measure and correlatesimilar metabolic changes when performing in vivo magnetic resonancespectroscopy for characterizing degree of disc degeneration.

HR-MAS spectroscopy was applied to intervertebral discs spanning a rangeof Thompson grades in order to identify the NMR observable chemicals andto determine the difference in the ratios of these chemicals betweendiscs at different stages of degeneration. Relaxation time measurementswere also performed to characterize changes in the environment ofchemical disc constituents with disc degeneration and their moleculardegrees of freedom.

2. Materials and Methods

A. Tissue Acquisition

This study was approved by our Institutional Review Board. Lumbar spineswere surgically removed from n=17 human cadavers (range: 22 to 85 years)and frozen at −80° C. The harvested spines were then separated with anautopsy saw and scalpel. The surrounding bone of the intervertebral bodywas removed and separated from the intervertebral disc. 3 mm biopsypunches were taken in the annulus fibrosus and nucleus pulposus regionsof the removed discs. These punches were taken in close proximity to oneanother and were cylindrically symmetrical. The average mass was15.2±3.4 mg. Three side-by-side samples, from a given location, werealso used to test for spectral reproducibility. The Thompson Grading wasperformed in consensus readings with adherence to the Thompson Gradingscale. Altogether, 8 Thompson grade 1, 6 Thompson grade 3 and 6 Thompsongrade 5 samples were studied.

B. HR-MAS Data Acquisition

HR-MAS data were acquired at 1.0±0.5° C. and a 2,250 Hz spin rate usinga Varian INOVA spectrometer operating at 11.75 T (500 MHz for ¹H) andequipped with a 4 mm gHX nanoprobe. For one-dimensional spectra, 40,000complex points were acquired over a 20,000 Hz (40 ppm) spectral width,with a 90° pulse width, 2 s HOD presaturaturation period, 32 transients,8 s repetition time (>5 times the longest T₁ relaxation time), 2 sacquisition time (>5 times the longest T₂ relaxation time), and a 3:36min total acquisition time. Samples were analyzed using custom designed18 μl zirconium rotors, containing an ellipsoid shaped sample chamberand an airtight screw top plug to prevent leakage. For each sample, 3.0μl of deuterium oxide containing 0.75 wt %3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid (D₂O+TSP, Sigma-Aldrich)were pipetted into the bottom of the rotor, after which the tissuesamples were weighed and then added.

Longitudinal (T₁) relaxation time measurements were acquired using aninversion recovery pulse sequence with variable delay times from 0.01 to2.00 s. Transverse (T₂) relaxation time measurements were acquired usinga rotor-synchronized (i.e., τ delay=n×(spin rate)⁻¹, where n is an evennumber) Carr-Purcell-Meiboom-Gill pulse sequence with echo times rangingfrom 10 to 128 ms. For two dimensional TOCSY spectra, 4096 complexpoints were acquired over a 20,000 Hz spectral width in the directdimension (F₂), while 256 complex points were acquired over a 6,500 Hzspectral width in the indirect dimension (F₁). TOCSY spectra wereacquired with a 2 s HOD presaturation/relaxation delay, 0.2 sacquisition time, 32 steady state pulses (1st increment only), 16transients/increment, mixing times ranging from 10 to 80 ms, phasesensitive using States-Habercorn, for a total experiment time ofapproximately 5 hrs, 12 min. To minimize the effects of B₀ and B₁inhomogeneities, rotor-synchronized constant adiabaticity WURST-8adiabatic pulses (33) were used for isotropic mixing, and were generatedusing the “Pandora's Box” pulse shape generator (Pbox, Varian) with a B₁field of 6,500 Hz and duration of 444 ms (1/spin rate). One-dimensionalspectra were acquired before and after each two-dimensional experimentto assess metabolic degradation. T₁ and T₂ relaxation time measurementswere taken from the nucleus (n=9) and the annulus (n=12) of healthy anddegenerate discs.

C. Data Processing

Data were processed online using Varian VNMR 6.1C software (Varian,Inc., Palo Alto), or offline using ACD/Labs 1D and 2D NMR processingsoftware, version 7.0 (Advanced Chemistry Development, Inc. Toronto).One dimensional FIDs were apodized with an exponential function, with aline broadening factor equal to the inverse of the acquisition time,Fourier transformed, phase corrected, and referenced to TSP at 0.0 ppm.Relaxation times were calculated using exponential least squaresregression analysis. Relaxation times were only used if the list (least)squares fit had a standard error of less than 10%. TOCSY data wereprocessed using 3×N linear predictions in F₁, zero filled to 1024complex points (F₁ only), and apodized using Gaussian weighting in bothdimensions.

Cross peaks were assigned using previously reported chemical shiftvalues from the literature. Based upon visual assessment of the data,three spectral regions from the 1D data were binned as follows: theN-acetyl region (1.90-2.10 ppm); the choline head group (Cho) region(3.15-3.30 ppm); and the carbohydrate (Carb) region (3.50-4.20 ppm).Three ratios, abbreviated N-Acetyl/Cho, Cho/Carb, and N-Acetyl/Carb,were then calculated for each spectrum, after setting the integratedarea of the carbohydrate region to 1.00. For each Thompson grade, themean ratios and standard deviations were calculated and a Student's ttest was performed to determine the statistical significance of thedata, where a p-value <0.05 was considered significant.

3. Results

A. Thompson Grade Differentiation

Representative one-dimensional HR-MAS spectra of the annular and nuclearregions of intervertebral discs with Thompson grades 1, 3, and 5 areshown in FIGS. 1-3.

FIGS. 1X, 1Y, 1Z show a representative 1-D HR-MAS spectra acquired froma Thompson Grade 1 disc (FIG. 1X) from the annulus fibrosus region(spectra at FIG. 1Y) and the nucleus pulposus region (spectra located atFIG. 1Z). Arrows illustrate associations between the various spectrashown at FIGS. 1Y, 1Z and the respective representative portion(s) ofthe disc being evaluated and shown at FIG. 1X. The circles indicate therepresentative location of 3 mm punch biopsies taken from the disc.Resolvable peaks include: A:isoleucine, leucine, valine; B:lactate,isoleucine; C:alanine; F:N-Acetyl resonance of chonroitin sulfate;G:glutamine; J:lysine; K:choline; L:phosphocholine; the bracketed regionindicates the C-H resonances of chondroitin sulfate.

Thompson grade 1 disc material is characterized by its stiff pliableannular ring and hydrated gel core (FIG. 1X). 3 mm punch biopsies weretaken from annular and nuclear regions of intervertebral discs and thecorresponding HR-MAS spectra are shown (FIG. 1Y, 1Z, respectively). BothHR-MAS spectra demonstrate a large N-acetyl resonance centered at 2.04ppm, and carbohydrate resonances attributed to chondroitin sulfate inthe region from 3.5 to 4.0 ppm. Additionally, resonances due to lactate(1.33 ppm), lipid (ppm), the choline head group (3.21-3.25 ppm), andseveral amino acids (alanine (1.49 ppm), isoleucine, leucine, andvaline) are also observable. Interestingly, in lower Thompson gradediscs, consistently greater spectral resolution was observed in thenucleus (FIG. 1Z) as compared to the annulus (FIG. 1Y).

FIGS. 2X, 2Y, 2Z show a moderately degenerated Thompson Grade 3 disc(FIG. 2X) and corresponding HR-MAS spectra taken from the annulusfibrosus (FIG. 2Y) and the nucleus pulposus (FIG. 2Z). Morphologicalchanges associated with Thompson grade 3 are dehydration of the disccoupled with a mechanical disruption of the disc matrix.Spectroscopically, the nucleus and annulus of Thompson Grade 3demonstrate an increase in spectral resolution in the carbohydrateregion of the spectrum compared to Thompson Grade 1 disc (FIG. 1X, 1Y,respectively). There is also an increase in the resonances containingthe choline headgroup (3.21 ppm).

Thompson grade 5 discs (FIG. 3X) pathologically demonstrate a furtherdehydration of the disc, mucinous infiltration and extensive disruptionsin the annulus, fibrous tissue replacement of the nucleus pulposus, anda loss of visual distinction between the annular and nuclear regions.Spectroscopically, there is a further increase in the resolution andintensity of the resonances in the choline and carbohydrate regions ofthe spectra. There is also a visual decrease in the intensity of theN-acetyl resonance and an increase in the number and intensity ofresonances due to free amino acids.

Table 1 shows integral ratios for annulus fibrosus (A) and nucleuspulposus (B) and student's t-test results. Individual and mean±sdevN-Acetyl/Cho, Cho/Carb, and N-Acetyl/Carb ratios for Thompson grades 1,3, and 5 discs are statistically compared. Thompson grades 2 and 4 discswere omitted from this study due to the subjectivity of the Thompsongrading scale. For both the nucleus and annulus, the mean N-Acetyl/Cho,and Cho/Carb ratios showed significant differences between all threeThompson grades, while the N-Acetyl/Carb ratio was only significantlydifferent between nucleus samples taken from Thompson 3 versus 5 discs.

For grades 1 vs. 3 the difference of ratios 1 and 2 were significantwhen comparing any two grades, for both the annulus and nucleus.N-Acetyl/Carb was only significant when comparing grade 1 to grade 5 ofthe nucleus. N-Acetyl/Cho=Integral (1.90-2.10 ppm)/Integral (3.15-3.30ppm), Cho/Carb=Integral (3.15-3.30 ppm)/Integral (3.50-4.20 ppm),N-Acetyl/Carb=Integral (1.90-2.10 ppm)/Integral (3.50-4.20 ppm).

In FIGS. 4A-4D, individual N-Acetyl/Cho (FIGS. 4A and 4B), and Cho/Carb(FIGS. 4C and 4D) ratios from the annulus (top) and nucleus (bottom) forthe three Thompson grades are plotted in order to assess the overlap ofindividual measurements. For the N-Acetyl/Cho ratio, there was nooverlap of individual nucleus values between Thompson grade 1 and 5discs, there was substantial overlap of individual values for all othercomparisons. For the Cho/Carb ratio, there was no overlap of individualannulus values between all three Thompson grades and no overlap ofindividual nucleus values between Thompson grade 1 and 5.

B. Total Correlation Spectroscopy (TOCSY)

To assign the resonances observed in the one-dimensional proton spectra,two-dimensional TOCSY spectra were acquired and the chemical shifts ofthe crosspeaks observed were compared to previously reported chemicalshift values. FIG. 5A shows a TOCSY spectrum of a Thompson grade 1intervertebral disc. In all eight of the Thompson grade 1 discs studied,only a limited number of crosspeaks could be observed, including thosedue to alanine (1.49, 3.79 ppm), lactate (1.35,4.16 ppm), and theprotons related to the carbohydrate portion of the proteoglycanpolymers. In contrast, the TOCSY spectrum of the six degeneratedThompson grade 5 discs studied (FIG. 5B), exhibited many more detectablecrosspeaks, including isoleucine (0.92, 1.32 ppm), leucine (0.98,1.72ppm), lysine (1.73, 3.04 ppm), proline (1.73, 1.93 ppm), glutamine(2.14, 2.46 ppm and 2.14, 3.79 ppm), glutamate (2.1, 2.36 ppm),hydroxyproline (2.45, 3.45 ppm), and ethanolamine (3.15, 3.83 ppm).TOCSY experiments demonstrated that the resolvable resonances in thecarbohydrate region (3.5-4.2 ppm) of the 1-D HR-MAS spectrum werecomposite peaks arising from multiple amino acids, ethanolaminecontaining compounds, as well as the sugar C-H protons of the breakdownproducts of chondroitin sulfate. The 1-D HR-MAS spectra of all discsstudied exhibited two singlets at 3.21 and 3.23 ppm, which correspond tothe chemical shifts of free choline (Cho) and phosphocholine (PC)respectively. TOCSY experiments also demonstrated cross peaks for themethylene protons of Cho at 3.55×4.07 ppm and PC at 3.62×4.18 ppm. Thereare also several other smaller, broader resonances in the choline regionof the spectrum, which remain unidentified.

C. T₁ and T₂ Relaxation Times

FIGS. 6A and 6B, respectively, show the average T₁ and T₂ relaxationtimes for resolvable resonances in the Thompson Grade 1, 3, and 5 discs.Only T₁ and T₂ relaxation times of resonances that could be resolved innucleus and annulus spectra of all of the Thompson grades were measured.This resulted in the measurement of T₁ and T₂ relaxation times forresonances at 0.9, 1.32, 1.49, 2.04, 3.21 and 3.67 ppm. There was nosignificant difference observed between T₁ and T₂ relaxation timesmeasured in the nucleus and annulus, therefore the relaxation times oftheses regions were combined to increase the statistical significance ofthe disc grade comparisons. The average T₁ and T₂ measurements for theseresonances from each of the Thompson grades demonstrated largevariability yielding no significant trend in metabolite T₁ and T₂ withincreasing disc degeneration. However, there was an observable trend forT₁ relaxation times with increasing Thompson grade. The observable trendfor T₁ values was an initial decrease for Thompson grade 3 discs andsubsequent increase for Thompson grade 5 discs. For T₂, there was noconsistent trend for all of the observed resonances, however, there wasan increasing trend for the N-Acetyl (2.04 ppm), Choline (3.21 ppm) andthe carbohydrate C-H resonances (3.67 ppm) with increasing Thompsongrade.

4. Discussion

Proton HR-MAS spectra were very similar for samples taken from annularand nuclear regions of intervertebral discs, with both spectrademonstrating a large N-acetyl resonance and carbohydrate resonancesprimarily from chondroitin sulfate, as well as resonances from cholinecontaining compounds, lipid/lactate and several amino acids. Due to themore gel like nature of the nucleus, consistently greater spectralresolution was observed in the nucleus as compared to the annulus forThompson grade 1 discs.

A substantial result observed in this study was that significant,visually apparent changes were observed in the proton HR-MAS spectra ofthe annular and nuclear samples from discs with increasing Thompsongrade. Specifically, there was a grade dependent increase in number ofobservable resonances and a sharpening of line widths of resonances inthe 3.5-4.0 ppm region of the spectrum, corresponding to a loss of the“broad component” in this region. Additionally, there was an increase inthe signal intensity of resonances in the choline containing compoundregion of the spectrum, and a relative decrease in the N-Acetylresonance.

Similarly, the number and intensity of cross peaks in TOCSY disc spectraincreased with increasing Thompson grade. A large number of the crosspeaks appearing in TOCSY spectra of degraded nucleus and annulus weredue to the amino acids hydroxyproline, proline, glycine, lysine,leucine, isoleucine, alanine, valine, glutamine and glutamate andethanolamine, many of which are components of collagen. Amino acidresonances also dominate the 1-D proton HR-MAS spectra of both nucleusand annulus in Thompson grade 5 discs. The observation of amino acidsdue to the breakdown of collagen has been previously observed in NMRstudies of cartilage digestion using metalloproteinases.Metalloproteinases (MMPs) have been suspected to play an important rolein disc degeneration by disrupting the collagen matrix that supports thedisc. As the collagen network disintegrates and the collagen helicesbreak down into their constituent amino acids, those resonances becomemore visible in both 1D and 2D HR-MAS spectra.

There was also metabolic evidence of chondroitin sulfate breakdown withdisc degeneration. This conclusion is based on the increase in intensityand resolution of carbohydrate C-H resonances (3.5-4.0 ppm) and therelative reduction in the N-Acetyl resonance of chondroitin sulfate(2.04 ppm) in proton HR-MAS spectra taken from the annulus and nucleusof Thompson grade 3 and 5 discs as compared to Thompson grade 1. Prior¹H and ¹³C HR-MAS NMR investigations on native and enzymaticallydigested bovine nasal cartilage have shown a change in the compositionof the N-acetyl resonance from being initially the N-acetyl resonance innon-digested cartilage to a composite peak containing the N-acetyl andamino acid resonances. Chondroitin sulfate concentration decreases withdisc degeneration. TOCSY studies have demonstrated that the resolvableresonances in the 3.5-4.0 ppm region of degenerated disc spectra alsoarise from a complex mixture of compounds including multiple aminoacids, ethanolamine containing compounds and the C-H resonances ofcarbohydrates. The complete assignment of resonances in degenerated diskspectra will therefore require human disc digestion studies andcorrelation with biochemical assays for chondroitin sulfate (e.g.dimethylmethylene blue (DMMB) assay) and collagen (collagenase).

Based on the observed changes in the N-Acetyl resonance, and resonancesin the choline and the carbohydrate/amino acid regions of the HR-MASspectrum, the N-Acetyl/Cho, Cho/Carb, and N-Acetyl/Carb ratios wereinvestigated to determine which ratios provided the best discriminationof Thompson grade. Both the mean N-Acetyl/Cho, and Cho/Carb ratiosshowed significant differences between all three Thompson grades, withthe Cho/Carb ratio demonstrating the least overlap between individualvalues for all three Thompson grades.

The Cho/Carb ratio had no overlap between the three Thompson grades forspectra taken from the annulus and minimal overlap for spectra takenfrom the nucleus. This is in particular beneficial since, for in vivospectroscopy, spectra acquisition solely from either the nucleus orannulus of the disc should thus not be required in many circumstances.This is a benefit due both to their close relative proximity and signalto noise considerations.

The N-Acetyl/Cho ratio may also prove useful for in vivo spectroscopy ofdisc degeneration since the N-Acetyl resonance is the largest peak inthe Thompson 1 and 3 discs and it was reduced to level that was lessthan or equal to the choline and carbohydrate regions of the spectrum inThompson grade 5 discs. In fact, Thompson grade 5 spectra from both thenucleus and annulus can be visually separated from Thompson grade 1 and3 disc spectra based on the relative reduction of the N-acetyl peak tothe choline and carbohydrate regions.

In prior studies, investigators have studied water spin-lattice andspin-spin relaxation times in an attempt to characterize disc andcartilage degeneration. More hydrated tissue is known to have a longerwater T₁ and T₂ and are shortened with disc degeneration, presumably dueto tissue water loss (14). No prior reports correlate T₁ and T₂ changesof the disc degenerative products with Thompson grade.

In this study the average T₁ and T₂ measurements of the disc breakdownproducts demonstrated large variability. However, there was anobservable trend in T₁ relaxation times. The trend for T₁ values was aninitial decrease for Thompson grade 3 discs and subsequent increase forThompson grade 5 discs. The observed initial shortening of breakdownproducts T₁'s in Thompson grade 3 discs could be for example due to theloss of water with disc degeneration. The subsequent increase of T₁'s inThompson grade 5 discs could be for example due to an increase inmobility of the breakdown products as they are released from theproteoglycan and collagen matrices.

Regarding observed changes in T₂, no clear trend exists across all thebreakdown products. T₁ and T₂ measurements of both water and degradationproducts in larger numbers of degenerated discs would provide furtheruseful information to understand the relaxation times measured in thisstudy.

5. Summary

In summary, proton HR-MAS provides spectra that are very similar forsamples taken from annular and nuclear regions of intervertebral discs.Significant, visually apparent changes are observable in the protonHR-MAS spectra of the annular and nuclear samples from discs withincreasing Thompson grade. Quantitatively, both metabolite peak areasratios of the resonances in the N-acetyl to choline regions, and cholineto carbohydrate regions of the spectra are useful to discriminate discsof increasing Thompson grade with minimal overlap of individual ratios.Changes in T₁ and T₂ relaxation times of the chemical constituents ofdisc spectra do not mirror changes in water relaxation times previouslyreported for disc degeneration. Changes in relaxation times of thechemical constituents of disc spectra with increasing degenerationreflect both changes in dehydration of the disc and the degree ofbreakdown of the proteoglycan and collagen matrices with increasingThompson grade. In vivo modalities of NMR spectroscopy will be usefulfor detecting chemical changes associated with disc degeneration.

In addition to the foregoing, the following references are hereinincorporated in their entirety by reference thereto:

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EXAMPLE 2

1. Introduction

Conventional imaging methods of assessing the painful, degeneratedintervertebral disc generally focus solely on morphologic criteria.However, it is well-known that there is a poor correlation betweenmorphologic findings and patient symptoms. The goal of this in vitrostudy is to utilize quantitative high-resolution magic angle spinning(HR-MAS) NMR spectroscopy as a tool to accurately characterizebiochemical markers in disc specimens harvested from patients undergoingsurgery. Spectra from discs obtained from patients that underwentdiscectomy for back pain and those of a reference population, consistingof patients undergoing surgery for scoliosis, were compared in attemptsto identify biochemical signatures of painful disc degeneration.

2. Materials and Methods

Spectral data were acquired at 11.7T (500 MHz), 1° C., and a 2,250 Hzspin rate using a Varian INOVA spectrometer equipped with a 4 mm gHXnanoprobe. Disc tissue removed at surgery in patients with discogenicpain (n=6) and patients with scoliosis undergoing anterior and/orposterior spinal fusion (n=4) were studied using custom designed 35 ulrotors. Quantitative proton spectra were acquired for tissue samples(mean=14.28±2.91 mg) with D2O+0.75% TSP as a standard (Sigma-Aldrich,St. Louis, Mo.). A spin-echo rotor synchronizedCarr-Purcell-Meiboom-Gill (CPMG) pulse sequence (nt=128, at=2.0 s, TR=5s, echo time=80 ms) was acquired for each tissue sample. The lactateresonance (1.31 ppm, doublet), n-Acetyl resonance associated withproteoglycans (PG) (2.04 ppm, singlet), and collagen breakdown region(col) (3.30-4.00 ppm) were analyzed to compare disc specimens. Theseregions are annotated in further detailed spectra sections shown inFIGS. 7A and 7B.

3. Results

FIGS. 7A, 7B show representative 80 ms ¹H CPMG spectra of (a) discogenicpain patient and (b) patient with scoliosis. The proteoglycan n-Acetylresonance (PG) lactate, and collagen breakdown region (col) areindicated.

Relative to deformity patients, those with back pain demonstratesignificantly lower PG/Lactate and PG/col ratios (p<0.05; see FIG. 7).In addition, Table 2 shows a table of information related to theexperiment performed that produced the exemplary spectra shown in FIGS.7A, 7B.

4. Discussion

The results from this experiment indicate that biochemical markers areuseful to characterize processes that correlate with discogenic pain.Previous studies report the influence of pH on proteoglycan synthesisand overall health. As lactate concentrations increase, the effective pHof disc material decreases due to the increase in free H+ in solution,which can hinder proteoglycan synthesis.

The direct causal relationship between lactate concentration and pain ispreviously unknown or explained in fine biochemical detail here withrespect to the present Experiment. However, the beneficial use oflactate concentration in providing a statistical correlation to pain isdemonstrated according to the methods performed and summarized here.This presents a substantially useful tool in diagnosing locality ofpain, regardless of mechanism of physical correlation between the twoparameters. The highly beneficial systems and methods herein describedprovide distinct benefit in allowing a non-invasive tool to correlatemeasured factors to pain, regardless of the particular biological“cause-and-effect” chemical or biological relationships underlying theseresults. Nonetheless, it is believed that increased lactate maystimulate nerve fibers in granulation tissue associated with dischealing. Further studies with larger numbers of clinically-relevantsamples that are matched for degeneration stage may be conducted by oneof ordinary skill based upon a review of this disclosure and otheravailable information, and to further confirm these and other areas ofinterest in identifying and using spectroscopic markers for assessingbiochemical degeneration and association with discogenic pain.

The following documents are herein incorporated in their entirety byreference thereto:

Keshari K R, Zektzer A S, Swanson M G, Majumdar S, Lotz J C, KurhanewiczJ. Characterization of intervertebral disc degeneration byhigh-resolution magic angle spinning (HR-MAS) spectroscopy. Magn ResonMed 2005; 53(3):519-527.

Maroudas A. The Biology of the Intervertebral Disc. In: Ghosh P, editor.The Biology of the Intervertebral Disc. Volume 2. Boca Raton: CRC Press;1988. p Ch. 9.

Urban J P, Smith S, Fairbank J C. Nutrition of the intervertebral disc.Spine 2004; 29(23):2700-2709.

It is to be appreciated based upon the foregoing disclosure that NMRspectroscopy is useful to identify and characterize spinal disc materialas to a corresponding degree of intervertebral disc degeneration, and inparticular with direct and predictable, reproducible correlation toThompson grades between discs. Accordingly, this represents one highlybeneficial, and broad aspect of the present invention. One particularembodiment described in fine detail hereunder relates to use of highresolution magic angle spinning (HR-MAS) spectroscopy, shown inparticular useful for explanted disc material observed in thatdiagnostic environment. However, other further, also highly beneficialembodiments also result, and represent further broad aspects disclosedhereunder, in regards to differentiating properties of living tissue invivo. Such may be accomplished for example, either using other types ofmagic angle spinning systems specially adapted for use with livingspecimens, or by use of other NMR spectroscopy systems useful onpatients and based upon suitably modified and adapted aspects and modesof the tools and methods taught hereunder.

By isolating high signal peaks for diagnostic pain correlation, as hasbeen done here for example in Example 2 summarized above, suchparticular targets are considered to extend well from 11.7T MAS MRItools and into equipment used directly with patients in clinicalpractice, e.g. 3 or 1.5T MRI equipment more typically used in clinicaldiagnosis. This may in particular be the case in the additionalapplication of customized local coils for creating higher local fieldsalong a region of interest, such as a particular region of lumbar spinefor example.

Still further, it is to be appreciated that tissue samples may be takenfrom patients, such as through biopsies, and then run in laboratoryequipment such as high field MRI machines, e.g. MAS NMR at 11.7T, foruseful patient diagnosis according to the various systems and methodsherein exemplified by way of the examples and description provided.

In addition, various exemplary chemicals and/or certain constituentfactors thereof are herein described as targets of non-invasivediagnosis of medical conditions associated with tissues. It isappreciated that such chemical “factors” may include the identifiedchemical or molecular structure itself, or a portion thereof, or ametabolite, degradation product, or bi-product thereof to the extent

SF-2005-063-5 36 correlative to the chemical identified. Moreover, thepresent disclosure deals with information that is produced by diagnostictools and methods to indicate certain property(s) of tissue. Suchproperty(s) may include for example pain or tissue degenerationthemselves, respectively. Or, it may include another second propertyhaving correlation or causal link with such first property. For example,nociceptive nerves, related growth factors, certain types ofinflammation, etc. may have causal links to either or both of pain andtissue degeneration. These may be the property directly indicated by theinformation produced by the present embodiments, whereas that indicatedproperty further leads to additional useful diagnosis and conclusion asto the related pain or degeneration. It is also contemplated that painand degeneration may be isolated results or targets of such diagnostictools and methods herein described, and may furthermore be linkedtogether in a combined result or target. Furthermore, degrees of suchproperties may be identified by the novel systems and methods hereindescribed. This may lead to further results and conclusions as tospatial relationship of such property within a tissue, e.g. the locationof a disc level, or portion of a disc (or other tissue structure), thatis more painful or degenerated relative to other surrounding joints,levels, or areas of tissue. Such localization may be the nature of theuseful information produced itself, or may be identified by furtheranalysis and processing conducted upon the useful information produced.

EXAMPLE 3

1. Introduction

The goal of this study is to extend prior experience, using quantitativehigh-resolution magic angle spinning (HR-MAS) NMR spectroscopy toaccurately characterize biochemical markers in disc specimens harvestedfrom patients undergoing surgery, to experience observing NMR spectra ofsimilar chemical signature targets in discs preserved in larger anatomicspecimens using a commercially available clinical MRI system andcommercially available surface coils. NMR spectroscopy data to beacquired and evaluated was for discs of ex-vivo bovine and cadavericspines positioned in commercially available head coils, and discs of aliving patient using a commercially available surface-spine coil. Papainwas injected into bovine discs studied. This was intended to inducechemical degradation of proteoglycan, in order to monitor change overtime in related magnetic resonance signals, in order to observedifferences in the related NMR spectral peaks using the clinical testequipment and methods.

2. Materials and Methods

All MRI examinations were performed on a 3 Tesla (3T) GE Excite Signawhole-body MR scanner. Spine samples were acquired for evaluating targetintervertebral discs in bovine (n=4) and cadaveric (n=4) ex-vivostudies. The ex-vivo studies used a GE 8-channel transmit/receive (T/R)phased array (PA) head coil or a GE 8-channel T/R PA knee coil. Thein-vivo patient evaluation was done using a GE 6-channel spine coil.Single voxel spectroscopy imaging was conducted using a short-echopoint-resolved spectroscopy (PRESS) sequence (TE/TR=35/2000 ms, 256repetitions, 1024 data points), including chemical shift-selected(CHESS) water suppression. Data analysis included combining the datafrom multiple channels and calculating the ration of spectroscopy peakheight of certain targets to be evaluated, namely N-Acetyl, Choline(Cho), and carbohydrate (carb), and additional data was evaluated forLactate (Lac).

For the bovine disc portion of the study, the discs were scanned beforePapain injection, 4 hours and 4-5 days after papain injection. Thepapain injection was made to achieve the enzymatic degradation of thenuclear pulpulsos. The solution was made by following the methoddescribed by Bradford (Spine 9:135-147).

The disc regions evaluated in the study are shown via voxel box overlayon MRI images of: a first bovine disc in FIGS. 8A, 9A, 10A, and 11A, ofa second bovine disc in FIGS. 12A, 13A, 14A, and 15A; five cadavericdiscs in FIGS. 16A, 17A, 18A, 19A, and 20A; respectively, and in-vivopatient discs in FIG. 21A. NMR spectra corresponding with each of theseimages, annotating the target peak spectral sections used for spectralevaluation, are shown in following corresponding FIGS.: 8B, 9B, 10B,11B, 12B, 13B, 14B, 15B, 16B, 17B, 18B, 19B, and 20B, respectively.Various Data Tables are provided for various peak ratios evaluated forthe specimens in the study, as noted below.

3. Results

A. Ex-Vivo Bovine Spine Study

For bovine disc #1 in the study, the T2-weighted images acquired at timeintervals (a) before Papain injection, (b) 4 hours, (c) 24 hours, and(d) 4-5 days after papain injection, are shown in FIGS. 8A, 9A, 10A, and11A, with single voxel overlay shown for disc nucleus region of the NMRspectral image taken. The corresponding spectroscopy images taken atthese time intervals are shown in FIGS. 8B, 9B, 10B, and 11B,respectively, with respective target peak regions evaluated in dataanalysis indicated by arrows on overlay. As shown in Table 3, dataanalysis for bovine disc #1 demonstrated that N-Acetyl/cho ratiodecreases with time and cho/carb composite ratio increases with time.Since papain injection is believed to cause disc degeneration, theresults agree with prior results of previous experiments comparingnon-degenerated vs. degenerated ex-vivo human disc tissue samples usingHR-MAS.

For bovine disc #2 in the study, the T2-weighted images acquired at timeintervals (a) before Papain injection, (b) 1-2 hours after injection,(c) 24 hours after injection, and (d) 4-5 days after injection, areshown in FIGS. 12A, 13A, 14A, and 15A, with single voxel overlay shownfor disc nucleus region of the NMR spectral image taken. Thecorresponding spectroscopy images taken at these time intervals areshown in FIGS. 12B, 13B, 14B, and 15B, respectively, with respectivetarget peak regions evaluated in data analysis indicated by arrows onoverlay. The 1-2 hour time interval data was not included in analysis asit is unknown whether 1-2 hours is enough time for papain to havesignificant effect on the disc tissue structures under evaluation. Asshown in Table 4 for this bovine disc #2 of the study, the N-Acetyl/choratio substantially decreases, and the cho/carb composite ratiosubstantially increases, over the time associated with tissuedegeneration in the disc sample. This confirms the experience withbovine disc #1 in the study, and the prior experience of previousstudies using similar peak ratio comparisons between non-degenerated anddegenerated ex-vivo human disc tissue samples using HR-MAS spectroscopy.

For bovine disc samples #3 and #4, only tabular results are shown of thevarious spectral peak ratios, in Tables 5 and 6, respectively. For thesediscs, another time point of 2 days after papain injection was added tofurther evaluate the experimental papain effects. At this time point,changes in the spectral ratios are not substantial as longer timepoints. However, as of the 5 day time point, the N-Acetyl/cho andCho/carb peak ratios for these bovine disc samples #3 and #4 areobserved to undergo substantial changes similar to those observed forbovine disc samples #1 and 2 in the study.

B. Ex-Vivo Cadaveric Spine Study

A cadaveric disc study was performed as follows. Five discs weretargeted for use from five cadaveric spine samples. For the firstcadaveric disc sample (reflected in T2-weighted image and single voxelspectral image in FIGS. 16A and B, respectively) this sample had a verylow MRI signal and was excluded in the data analysis. Therefore, onlythe other four cadaveric discs intended for study were included in theanalysis, indicated as cadaveric disc samples #1-4. T2-weighted imagesfor these cadaveric discs #1-4 included in the analysis for this studyare shown in FIGS. 17A, 18A, 19A, and 20A, with single voxel overlayshown for the disc nucleus region of NMR spectral image taken. Thecorresponding spectroscopy images taken at these time intervals areshown in FIGS. 17B, 18B, 19B, and 20B, respectively, with respectivetarget peak regions evaluated in data analysis indicated by arrows onoverlay. The number designation given to the test samples 1-4 werechosen in reverse order as to degree of their respective degradation asdetermined structurally per the T2-weighted MRI images (eg. Disc 1 beingthe most degradated, and Disc 4 being the least degradated in the samplepopulation).

Table 7 shows similar spectral peak ratios analyzed for the cadavericspine study as prior studies noted above. As indicated by bolded arrowsin the first column, certain trends were apparent in each peak ratiopresented between most to least degenerated discs per T2-weighted MRIimage analysis. In particular, similar trends of N-Acetyl/cho andCho/carb peak ratios were observed for the ex-vivo cadaver spine studyas with prior bovine spine experience and prior HR-MAS disc tissuesample experience. More specifically, N-Acetyl/cho peak ratios weresubstantially lower, and below 1 indicating choline peak larger thanN-Acetyl peak, for the more degradated Disc 1 and trending upward toN-Acetyl peak representing several multiple value to the choline peakfor the most degradated Disc 4 among the samples. Similarly, thecho/carb peak ratios were substantially higher for the more degradateddiscs than the lower degradated discs, and in particular at the extremescomparing the respective data for Discs 1 and 4.

C. In Vivo Human Patient Study

A healthy patient volunteer executed informed consent to NMRspectroscopy data acquisition for purposes of this study during an MRIscan taken of the patient's spine for other purposes. A T2-weightedimage for the patient's spine is shown in FIG. 21A, with single voxeloverlay shown for the disc nucleus region of NMR spectral image taken.The corresponding spectroscopy image is shown in FIG. 21B, withrespective target peak regions evaluated in data analysis indicated byarrows on overlay. Based upon the T2-weighted image, and absence ofclinical symptoms associated with degeneration or pain, the disc imagedis considered to be representative sample of a relatively normal,healthy disc.

Table 8 shows the similar peak spectral ratios for this in-vivo patientdisc as those analyzed in prior studies noted above. As illustrated inthe NMR spectrum shown in FIG. 21B, the single voxel region imaged inthe disc nucleus (box overlay in FIG. 21A) did not appear to produce arepresentative choline (cho) peak in the NMR spectrum imaged for theregion. Accordingly, as shown in Table 8, the N-Acetyl/cho peak ratio isshown as infinity, as the denominator is zero according to the absenceof data in the NMR spectral data. Similarly, the cho/carb ratio isreflected in Table 8 as 0, again due to the absence of choline thus azero value given to the absent peak.

The absence of choline is generally expected for typical normal healthydiscs, as we have previously demonstrated that choline levels increasewith disc degeneration. Choline is typically found in lipids that makeup cell membranes and in the neurotransmitter acetylcholine.Consequently, the elevated choline levels noted in severely degenerateddiscs may represent increased cellularity associated withmicrovascularization and innervation, as compared with typical normalhealthy discs. Thus, it is expected that a trend in NMR spectroscopyevaluation of normal healthy human discs should reveal the resultsreflected in the data shown for this pilot patient study. Based on ourprior study with cadaveric tissues, there is an increasing presence ofcholine found in the NMR spectra imaged in increasingly degenerateddiscs, reflected in significantly decreased N-Acetyl/cho peak ratios andincreased cho/carb peak ratios.

4. Discussion

The results from the study components included in this experimentconfirm expected results of prior experiment conducted onnon-degenerated and degenerated human disc tissue samples using HR-MASspectroscopy. Trends in ratios between certain readily identifiable NMRspectral peaks known to represent N-Acetyl (generally associated withproteoglycan presence), choline (generally associated with cellularity),and carbohydrate, are confirmed to predictable outcomes in evaluatingdegeneration and as frequently associated with pain. The data analyzedin this study indicate that these peak ratios, and corresponding trends,can be readily evaluated using clinical MRI and local coil equipment.Each of four bovine spines evaluated in 3T MRI systems with local headand knee coils reflected similar identifiable changes in N-Acetyl/choand cho/carb peaks under experimentally induced conditions believed toreasonably simulate general biochemical changes in the discs over adegenerative process. The one in-vivo human patient experience using a3T MRI system and local spine coil reflected a predictable NMR spectralimage of a normal healthy disc with respect to these particular peakratios. This clinical experience confirms expected results from theprior ex-vivo experiments previously conducted.

5. Conclusion

The prior results analyzing NMR spectra of ex-vivo human spinal disctissue samples using HR-MAS spectroscopy indicated that NMR spectralpeak ratios associated with N-Acetyl/cho and cho/carb, and changes inthose ratos, demonstrate repeatable and predictable characteristicsbetween generally normal healthy discs and degenerated discs.Experiments conducted on whole bovine and cadaver spine specimens usingclinical 3T MRI systems with commercially available local head and kneecoils confirmed these prior results. This confirming experience inlarger portions of anatomy (eg. whole preserved spine sections vs.excised disc tissue specimens) as test samples, using commerciallyavailable clinical MRI systems and local coils, further indicatesclinical utility of imaging disc degradation and pain via the NMRspectroscopy analysis presented. Further in-vivo study conducted on oneliving human patient using a clinical 3T MRI system and local spine coilfurther confirmed NMR spectroscopy results of these peak ratios expectedfor normal healthy discs according to the prior experience in theprevious ex-vivo studies, in particular in relation to N-Acetyl/cho andcho/carb peak ratios. Based upon this initial experience with one normalhealthy human disc, an expected increase in choline from such normalhealthy discs to degenerated discs would result in an expected decreaseand increase, respectively, in these ratios. Simple further dataacquisition and analysis from more patients with varying degrees of discdegradation per T2-weighted MRI may be readily performed and expected toconfirm the expected trends and utility of such analysis in patientdiagnosis and care. This further indicates that NMR spectroscopyconducted in the manner presented here has clinical utility innon-invasive imaging and evaluation of disc degeneration, including asis believed to be associated with pain. In particular, this studyconfirms clinical utility using non-invasive NMR spectroscopy with acommercially available clinical 3T MRI system and commercially availableclinical spine coil for determining localized extent of disc degradationin a spine. In particular, the NMR spectral peak ratios of N-Acetyl/choand cho/carb provide useful data in making this determination in what isbelieved to be a predictable, repeatable manner in a non-invasiveclinical diagnostic setting.

It is further appreciated that certain surface coils were evaluatedamong the studies conducted, with confirming results. Examples includehead, knee, and spine surface coils. It is further appreciated that moreinvasive approaches, such as for example probe-based coils, may providestill further benefit and utility when incorporated with the severalaspects of the present disclosure, including for example enhancedresolution and spatial sensitivity with increased signal to noise ratioswith respect to target NMR spectral features to be evaluated. Forexample, certain probe-based coils have been previously incorporated forprostate cancer evaluation in clinical MRI systems.

The present disclosure, to the extent directed toward specified systemsand devices of the embodiments, further contemplates respective methodsrelated thereto, whether or not such method(s) are specificallydescribed in detail aside from their contemplated use in the systemdisclosure. One of ordinary skill will understand such relationshipbased upon the totality of the disclosure provided herein. Similarly,methods disclosed hereunder further contemplate respective system anddevice aspects clearly contemplated by such disclosure, whether or notspecific reference to such system or device aspects is provided inparticular aside from the method description. The foregoing relates tothe description provided hereunder, as well as the claims providedbelow. For example but without limitation, it is to be appreciated thatcertain functional aspects (or interco-operation described betweenelements) of system or apparatus claims provided herewith furthercontemplate the methods of performing such function as additional,independent aspects contemplated hereunder, though not necessarily to beapplied as limitations to the particularly specified aspects and relatedmodes and embodiments unless described expressly so.

Although the description above contains many details, these should notbe construed as limiting the scope of the invention but as merelyproviding illustrations of some of the presently preferred embodimentsof this invention. Therefore, it will be appreciated that the scope ofthe present invention fully encompasses other embodiments which maybecome obvious to those skilled in the art, and that the scope of thepresent invention is accordingly to be limited by nothing other than theappended claims, in which reference to an element in the singular is notintended to mean “one and only one” unless explicitly so stated, butrather “one or more.” All structural, chemical, and functionalequivalents to the elements of the above-described preferred embodimentthat are known to those of ordinary skill in the art are expresslyincorporated herein by reference and are intended to be encompassed bythe present claims. Moreover, it is not necessary for a device or methodto address each and every problem sought to be solved by the presentinvention, for it to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed under the provisions of 35U.S.C. 112, sixth paragraph, unless the element is expressly recitedusing the phrase “means for.”

TABLE 1 Integrated Areas Grade 1 Grade 3 Grade 5 1 vs 3 1 vs 5 3 vs 5 A:Disc Metabolite Ratios, Annulus Fibrosus N-Acetyl/Cho 3.685 ± 0.6012.552 ± 0.339 1.941 ± 0.540 0.006 0.035 0.035 Cho/Carb 0.115 ± 0.0270.191 ± 0.014 0.243 ± 0.016 <0.001 <0.001 <0.001 N-Acetyl/Carb 0.420 ±0.137 0.487 ± 0.062 0.466 ± 0.096 0.277 0.486 0.638 B: Disc MetaboliteRatios, Nucleus Pulposus N-Acetyl/Cho 5.487 ± 1.400 4.336 ± 0.706 2.364± 0.411 0.031 <0.001 0.001 Cho/Carb 0.082 ± 0.018 0.121 ± 0.014 0.161 ±0.006 0.001 <0.001 0.001 N-Acetyl/Carb 0.448 ± 0.139 0.515 ± 0.049 0.380± 0.079 0.465 0.146 0.006

TABLE 2 Ratios of PG/lactate, PG/Col, and Lactate/Col + 1 std deviationDisc Pain Scoliosis PG/Lactate 0.37 ± 0.36 1.72 ± 0.81 PG/col 0.28 ±0.14 0.65 ± 0.35 Lactate/col 0.70 ± 0.35 0.38 ± 0.08

TABLE 3 Ex-Vivo Bovine Disc #1; NMR Spectral Peak Ratios over time(Papain Injection) NMR Peak Before 4 hrs 24 hrs 4 days Ratios Injectafter Inject after Inject after Inject N-Acetyl/Lac + Lip 

4.88 2.52 2.65 1.64 N-Acetyl/cho 

10.32 3.87 3.27 2.64 Cho/carb 0.31 0.43 0.53 0.82 N-Acetyl/carb 3.171.66 1.76 2.17

TABLE 4 Ex-Vivo Bovine Disc #2; NMR Spectral Peak Ratios over time(Papain Injection) NMR Peak Before 1-2 hrs 24 hrs 4 dys Ratios Injectafter Inject after inject after inject N-Acetyl/Lac + Li 

1.56 3.30 3.32 2.17 N-Acetyl/cho 

7.65 7.69 3.10 2.90 Cho/carb 0.26 0.20 0.48 0.66 N-Acetyl/carb 1.97 1.561.48 1.91

TABLE 5 Ex-Vivo Bovine Disc #3; NMR Spectral Peak Ratios over time(Papain Injection) NMR Spectral before 24 hrs 2 days 5 days Peak RatiosInjection after Inject after Inject after Inject N-Acetyl/ 8.47 7.655.16 3.35 Lac + Lip N-Acetyl/cho 

4.23 3.31 4.13 2.05 Cho/carb 

0.49 0.73 0.59 1.17 N-Acetyl/carb 2.07 2.42 2.45 2.40

TABLE 6 Ex-Vivo Bovine Disc #4; NMR Spectral Peak Ratios over time(Papain Injection) NMR Spectral Before 24 hrs 2 days 5 days Peak RatiosInject after Inject after Inject after Inject N-Acetyl/ 2.38 2.26 3.664.25 Lac + Lip N-Acetyl/cho 

8.50 6.39 7.60 2.08 Cho/carb 

0.17 0.54 0.33 0.77 N-Acetyl/carb 1.48 3.45 2.48 1.60

TABLE 7 Ex-Vivo Cadaver Discs #1-4; NMR Spectral Peak Ratios taken forDiscs 1-4 (in reverse order of degree of degradation per structuralanalysis in T2-weighted MRI, eg. Disc 1 most degradated, Disc 4 leastdegradated); Arrows indicate trend in peak ratio values from Disc 1 toDisc 4. NMR Peak Ratios Disc 1 Disc 2 Disc 3 Disc 4 N-Acetyl/ 0.21 0.330.76 2.26 Lac + Lip 

N-Acetyl/cho* 

0.48 0.87 2.96 17.28 Cho/carb* 

0.60 0.60 0.43 0.22 N-Acetyl/carb 

0.28 0.52 1.27 3.88

TABLE 8 In-Vivo Human Patient Spine NMR Spectroscopy Study NMR SpectralPeak Ratios Peak Ratio Values N-Acetyl/Lac + Lip 2.86 N-Acetyl/cho ∞(expected to decrease w/disc degeneration) Cho/carb 0 (expected toincrease w/disc degeneration)

1-68. (canceled)
 69. A medical diagnostic system for providingdiagnostic information from a nuclear magnetic resonance (NMR) spectrumgenerated and acquired from a region of tissue of a pain patient fordetermining if degenerative nociceptive pain is occurring in the regionof tissue, comprising: an NMR spectroscopy system in a configurationthat is operable to apply a magnetic pulse sequence to artificiallyinduce NMR signals in the region of tissue, and wherein the NMRspectroscopy system comprises a local detector coil assembly in aconfiguration that is operable, in response to the applied magneticpulse sequence, to acquire the NMR signals, to thereby generate NMRspectroscopic data related to an NMR spectrum from the region of tissueand to provide the NMR spectroscopic data related to the NMR spectrum ina form that is processable; a computer processor; a non-transitorycomputer-readable memory storing instructions in the form of a computerreadable software program executable by the computer processor toprocess the NMR spectroscopic data provided by the NMR spectroscopysystem, wherein the instructions, when executed by the computerprocessor, perform operations to: determine a ratio value based at leastin part on a ratio between spectral parameters associated with (a) ann-Acetyl-related resonance region of the NMR spectrum associated withchondroitin sulfate or a metabolite or degradation product thereof, and(b) a lactate-related resonance region of the NMR spectrum; and providediagnostic information at least in part based upon the ratio value, andwherein the diagnostic information corresponds with degenerativenociceptive pain.
 70. The system of claim 69, wherein the computerprogram is further executable by the computer processor to process theNMR spectroscopic data to determine the ratio value based at least inpart upon a measured feature of an n-Acetyl-related resonance region ofthe NMR spectrum.
 71. The system of claim 69, wherein the computerprogram is further executable by the computer processor to process theNMR spectroscopic data to determine the diagnostic information based atleast in part upon a collagen-related factor.
 72. The system of claim69, wherein the computer program is further executable by the computerprocessor to process the NMR spectroscopic data to determine thediagnostic information based at least in part upon a measured feature ofa choline-related region of the NMR spectrum.
 73. The system of claim69, wherein the computer program is further executable by the computerprocessor to process the NMR spectroscopic data to determine thediagnostic information based at least in part upon a measured feature ofa carbohydrate-related region of the NMR spectrum.
 74. The system ofclaim 69, wherein the computer program is further executable by thecomputer processor to process the NMR spectroscopic data to determinethe diagnostic information based at least in part upon a measuredfeature of a region of the NMR spectrum associated with acollagen-related factor that comprises a chemical entity indicative ofcollagen break-down in the region of tissue.
 75. The system of claim 69,wherein the computer program is further executable by the computerprocessor to process the NMR spectroscopic data to determine thediagnostic information based at least in part upon a first calculatedratio between a measured feature of the NMR spectrum associated with aproteoglycan-related factor and a measured feature of the NMR spectrumassociated with a lactate-related factor, and a second ratio between ameasured feature of the NMR spectrum associated with theproteoglycan-related factor and a measured feature of the NMR spectrumassociated with a collagen-related factor in the region of tissue. 76.The system of claim 69, wherein the NMR spectroscopy system comprises a3 Tesla NMR system.
 77. The system of claim 69, wherein the NMRspectroscopy system comprises: a single voxel region prescribed tocoincide with the region of tissue; wherein the NMR spectroscopy systemis operable to apply the magnetic pulse sequence to induce the NMRsignal at the single voxel region and acquire the NMR spectroscopic datafrom the single voxel region via the local detector coil assembly. 78.The system of claim 77, wherein the NMR spectroscopy system comprises:an additional single voxel region prescribed to coincide with anadditional region of tissue; wherein the NMR spectroscopy system isoperable to apply an additional magnetic pulse sequence to induce NMRsignal at the additional single voxel region and acquire an additionalNMR spectroscopic data from the second single voxel region via the localdetector coil assembly.
 79. The system of claim 69, wherein the at leastone computer program is further executable by the computer processor todisplay a curve related to the NMR spectrum, and a portion of the curvecomprises each the spectral parameter.
 80. The system of claim 69,wherein: respective spectral parameters of the lactate-related resonanceregion and the n-Acetyl-related resonance region correspond to alactate-related chemical factor and a proteoglycan-related chemicalfactor, respectively; and wherein the ratio value comprises a ratiobetween respective amounts of the chemical factors.
 81. The system ofclaim 69, wherein the region of tissue comprises an intervertebral discof a spine of a patient.
 82. The system of claim 69: wherein the NMRspectroscopy system is operable to apply a magnetic pulse sequence toartificially induce NMR signals in an additional region of tissue;wherein the local detector coil assembly is operable, in response to theapplied magnetic pulse sequence, to acquire the NMR signals, to therebygenerate NMR spectroscopic data related to an additional NMR spectrumfrom the additional region of tissue and to provide the NMRspectroscopic data related to the NMR spectrum in a form that isprocessable; wherein the computer program is further executable by thecomputer processor to determine an additional ratio value based at leastin part a ratio between spectral parameters associated with a) ann-Acetyl-related resonance region of the additional NMR spectrumassociated with chondroitin sulfate or a metabolite or degradationproduct thereof, and (b) a lactate-related resonance region of theadditional NMR spectrum; and wherein the computer program is furtherexecutable by the computer processor to provide the diagnosticinformation at least in part based upon the additional ratio value. 83.The system of claim 82, wherein the instructions, when executed by thecomputer processor, perform operations to compare the ratio value andthe additional ratio value, and wherein the diagnostic information isbased at least in part on the comparison.
 84. The system of 83, whereinthe diagnostic information comprises a determination of where thedegenerative nociceptive pain is being experienced between the region oftissue and the additional region of tissue based at least in part on thecomparison.
 85. A method for diagnosing whether local degenerativenociceptive pain is occurring in a region of tissue of a pain patient,the method comprising: analyzing at least a lactate-related resonanceregion and a proteoglycan-related resonance region from nuclear magneticresonance (NMR) spectroscopic data associated with a respective nuclearmagnetic resonance (NMR) spectrum generated via acquired NMR signalsinduced in the region of tissue via a magnetic pulse sequence appliedvia an NMR spectroscopy system that comprises a local detector coilassembly in a configuration that is operable, in response to the appliedmagnetic pulse sequence, to acquire the NMR signals and generate thespectroscopic data from the region of tissue positioned in the NMRspectroscopy system; producing a ratio value from the analysis for theNMR spectrum from the region of tissue and that indicates a ratiobetween spectral parameters associated with the lactate-relatedresonance region and the proteoglycan-related resonance region;determining whether degenerative nociceptive pain is being experiencedat the region of tissue based at least in part on the ratio value; andwherein the analyzing and the producing are performed by running acomputer software program on a computer processor.
 86. The method ofclaim 85, further comprising: configuring an NMR spectroscopy system ina configuration that is operable to apply the magnetic pulse sequence toinduce the NMR signals, acquire the induced NMR signals, and generatethe NMR spectroscopic data for the acquired NMR signals, from the regionof tissue; and acquiring the NMR signals and generating thecorresponding NMR spectroscopic data from the region of tissue byoperating the NMR spectroscopy system in the configuration.
 87. Themethod of claim 86, wherein the NMR spectroscopy system comprises a 3Tesla NMR system.
 88. The method of claim 85, wherein the region oftissue comprises an intervertebral disc of a spine of a patient.
 89. Themethod of claim 85, comprising determining whether degenerativenociceptive pain is being experienced at the region of tissue based atleast in part on a collagen-related factor.
 90. The method of claim 85,comprising determining whether degenerative nociceptive pain is beingexperienced at the region of tissue based at least in part on a measuredfeature of a choline-related region of the NMR spectrum.
 91. The methodof claim 85, comprising determining whether degenerative nociceptivepain is being experienced at the region of tissue based at least in parton a measured feature of a carbohydrate-related region of the NMRspectrum.
 92. The method of claim 85, comprising determining whetherdegenerative nociceptive pain is being experienced at the region oftissue based at least in part on a measured feature of a region of theNMR spectrum associated with a collagen-related factor that comprises achemical entity indicative of collagen breakdown in the region oftissue.
 93. The method of claim 85, comprising determining whetherdegenerative nociceptive pain is being experienced at the region oftissue based at least in part on a first calculated ratio between ameasured feature of the NMR spectrum associated with aproteoglycan-related factor and a measured feature of the NMR spectrumassociated with a lactate-related factor, and a second ratio between ameasured feature of the NMR spectrum associated with theproteoglycan-related factor and a measured feature of the NMR spectrumassociated with a collagen-related factor in the region of tissue. 94.The method of claim 85, further comprising displaying a curve related tothe NMR spectrum, and a portion of the curve comprises each the spectralparameter.
 95. The method of claim 85, further comprising: analyzing atleast a lactate-related resonance region and a proteoglycan-relatedresonance region from nuclear magnetic resonance (NMR) spectroscopicdata associated with a respective nuclear magnetic resonance (NMR)spectrum generated via acquired NMR signals induced in an additionalregion of tissue via a magnetic pulse sequence applied via the NMRspectroscopy system that comprises the local detector coil assembly in aconfiguration that is operable, in response to the applied magneticpulse sequence, to acquire the NMR signals and generate thespectroscopic data from the additional region of tissue positioned inthe NMR spectroscopy system; producing an additional ratio value fromthe analysis for the NMR spectrum from the region of tissue and thatindicates a ratio between spectral parameters associated with thelactate-related resonance region and the proteoglycan-related resonanceregion; and wherein the analyzing and the producing are performed byrunning a computer software program on a computer processor.
 96. Themethod of claim 95, comprising: comparing the ration value to theadditional ration value; and determining where the degenerativenociceptive pain is being experienced between the region of tissue andthe additional region of tissue based at least in part on thecomparison.
 97. A medical diagnostic system configured to providediagnostic information that is indicative of a property of a firstregion of tissue removed from a patient, comprising: a nuclear magneticresonance (NMR) spectroscopy system that is configured to generate NMRspectroscopic data related to an NMR spectrum of the first region and toprovide the NMR spectroscopic data related to the NMR spectrum in a formthat is processable to provide the diagnostic information; wherein theNMR spectroscopy system comprises a proton high resolution magic anglespinning (HR-MAS) spectroscopy system that is configured to produce theNMR spectroscopic data; a processor that is configured to process, basedon a set of encoded program instructions executable on the processor,the NMR spectroscopic data provided by the NMR spectroscopy system so asto provide the diagnostic information based at least in part upon ann-Acetyl-related resonance region of the NMR spectrum and alactate-related resonance region of the NMR spectrum; wherein theproperty comprises a chemical environment of the first region of tissue;and wherein the diagnostic information is correlative to pain.